Butterfly eyespots exhibit unique patterns of open chromatin.

How the precise spatial regulation of genes is correlated with spatial variation in chromatin accessibilities is not yet clear. Previous studies that analysed chromatin from homogenates of whole-body parts of insects found little variation in chromatin accessibility across those parts, but single-cell studies of brains showed extensive spatial variation in chromatin accessibility across that organ. In this work we studied the chromatin accessibility of butterfly wing tissue fated to differentiate distinct colors and patterns in pupal wings of We dissected small eyespot and adjacent control tissues from 3h pupae and performed ATAC-Seq to identify the chromatin accessibility differences between different sections of the wings. We observed that three dissected wing regions showed unique chromatin accessibilities. Open chromatin regions specific to eyespot color patterns were highly enriched for binding motifs recognized by Suppressor of Hairless (Su(H)), Krüppel (Kr), Buttonhead (Btd) and Nubbin (Nub) transcription factors. Genes in the vicinity of the eyespot-specific open chromatin regions included those involved in wound healing and SMAD signal transduction pathways, previously proposed to be involved in eyespot development. We conclude that eyespot and non-eyespot tissue samples taken from the same wing have distinct patterns of chromatin accessibility, possibly driven by the eyespot-restricted expression of potential pioneer factors, such as Kr.